During embryogenesis, the Notch/RBPjk and Wnt/2-catenin signaling pathways regulate diverse cell fate decisions. Osteoblasts are the primary cell type responsible for producing bone matrix and are necessary not only for bone development but also bone remodeling and homeostasis. Deficiency in osteoblast differentiation results in osteoporosis, a disease characterized by significant decreases in bone mass. During osteoblast development, Notch/RBPjk signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation. Conversely, Wnt/2-catenin signaling promotes osteoblast differentiation. The disparate effects of these two pathways are clear, with Notch inhibiting differentiation while Wnt promoting it. However, the exact stage at which Notch inhibits osteoblast development has not been defined, although our preliminary studies indicate that Notch likely impedes further progression of Runx2- positive cells. Moreover, the genetic relationship between Wnt and Notch pathways during osteoblast differentiation is not known. As such, the goal of this proposal is to test the hypothesis that Notch suppresses further differentiation of Runx2-positive cells and that Notch functions at least in part by inhibiting Wnt/ -catenin signaling. To test this hypothesis I have developed two specific aims. Aim 1. Determine the role of Notch/RBPjk signaling in Runx2-positive cells during osteoblast differentiation I will use a novel mouse line to delete RBPjk in Runx2-positive cells in a doxycycline-inducible manner and to assess the potential bone phenotype. This study will provide genetic evidence about the stage-specific regulation of osteoblast differentiation by Notch/RBPjk signaling. Aim 2. Evaluate the genetic relationship between RBPjk and -catenin during osteoblast development I will examine the effect of -catenin removal (single or both alleles) on the high-bone-mass phenotype caused by RBPjk deletion in mesenchymal progenitors. This study will test the hypothesis that Notch/RBPjk functions genetically upstream of Wnt/ -catenin signaling during osteoblast differentiation. Overall, these studies will enhance the current understanding about Notch inhibition of osteoblast differentiation, and shed light on the functional relationship between two opposing signals, Notch and Wnt, during osteoblast development. PUBLIC HEALTH RELEVANCE: Osteoporosis is a major public health problem affecting approximately 75 million individuals worldwide. Findings from these studies may lead to novel therapeutic approaches to the treatment of this disease.